Modern Applied Science June, 2009

Purification and Partial Characterization of Esterase from Marine Vibrio fischeri
P. Ranjitha (Corresponding author) Research Department of Microbiology, Sengunthar Arts and Science College Tiruchengode 637 205, Tamilnadu, India E-mail: E.S. Karthy Research Department of Microbiology, Sengunthar Arts and Science College Tiruchengode 637 205, Tamilnadu, India A. Mohankumar Research Department of Microbiology, Sengunthar Arts and Science College Tiruchengode 637 205, Tamilnadu, India

Lipolytic enzymes from marine microbes have been the focus of intense and growing research. The bioluminescence bacterium Vibrio fischeri was produced esterase enzyme when the medium contained specific substrate. The esterase was purified from the concentrated culture supernatant. The most active fractions were obtained using the technique of precipitation with 1N HCl. The precipitated fraction was purified by ion exchange chromatography (DEAE-Cellulose) and gel filteration chromatography (Sephadex G200). The purified active fraction exhibiting final specific activity of 300U/mg and characterized; the optimum pH was 7.5, the optimum temperature was 30°C. The enzyme was very stable at the temperature 30°C and at wide range of pH. The enzyme was monomeric protein having molecular mass of 37 KDa estimated by native PAGE assay.
Keywords: Vibrio fischeri, Lipolytic enzymes, Extracellular esterase 1. Introduction

Marine microorganisms which are salt tolerant, provide an interesting alternative for therapeutic purposes. Marine microorganisms have a diverse range of enzymatic activity and are capable of catalyzing various biochemical reactions with novel enzymes. Especially, halophilic microorganisms possess many hydrolytic enzymes and are capable of functioning under conditions that lead to precipitation of denaturation of most proteins. Further it is believed that sea water, which is saline in nature and chemically closer to the human blood plasma, could provide microbial products, in particular the enzymes, that could be safer having no or less toxicity or side effects when used for therapeutic applications to humans (Sabu, 2003). The Photobacterium (Vibrio) fischeri group consists of rod-shaped cells with a light yellow, cell-associated pigment and a tuft of sheathed flagella (Hendrie et al., 1970). The species is restricted to the marine environment and has a specific requirement for sodium ion for growth (Reichelt and Baumann, 1973). It occurs both free living in sea water (Ruby and Nealson, 1976) and as the specific luminous symbiont of the monocentrid fish and squid. Esterases are distinguished from lipases in that their action is generally restricted to short-chain fatty acids. Esterases catalysis of a large number of aliphatic and aromatic esters. Although the molecular and catalytic properties of this protein from mammalian sources have been well studied, only limited investigations have been made in to properties of isolated microbial esterases. Because of the potential food applications, the general economic attractiveness of extracellular enzyme is higher than the intracellular microbial industrial enzymes (Meghji et al., 1990).

1996). Esterase activities at different hours were compared by measuring the width (millimeter) of areas of cleaning or area of deep blue color around the colonies.) were collected and cooled to about 8ºC before opening the mantle cavity along the ventrum. 2.3. 2. 3. glycerol and ethanol. tween 20. The production medium was adjusted using a standard pH meter and incubated 30ºC for 24 hrs. 0.5 Time Course of Esterase Enzyme Production Lipolytic enzyme of esterase production was observed from the initial time of inoculation to decline phase of the V. fischeri isolated from squid and on the purification and partial characterization of its esterase.05g of CaCl2.5% peptone. Therefore. after solidification of these media inoculated and incubated at 30ºC for 10 days.. The main applications are in cosmetics.1 Bacteria and Growth Conditions The squids (Sepia sp. The following two methods were used to obtain samples of symbiotic luminescent bacteria.3% glycerol) agar plates and identified the luminous bacteria based on the work of Reichelt and Baumann (1973) and VHA (Vibrio Hareyi Agar) differential media (Harris et al. 5. 2.3. lauric acid). the objective of this study is to focus on the esterase production of V. 2.25% Triacylglycerol (tributyrin) were then poured into petri disches.3% yeast extract.1 ZoBell 2216E Modified Media All strains were precultivated on the solid maintenance SWC medium. 1992).38M NaCl. 42 and 72 hrs at 30 ºC.3. No.4 Different Carbon Sources A defined minimal salt medium was added with carbon sources included triacylglycerol (tributyrin). fischeri. After 1-10 day incubation the halos. 0. coconut oil) free fatty acids (oleic acid. 15 g of bacto agar made to 1 L with aged seawater.2 Screening of Esterase Enzyme 2. clear (on tributyrin) or turbid (on all other substrates) were measured.7H2O.6H2O.02M MgCl2. The total diameter. 24. It contains l0g of peptone. pH was adjusted to 7.Vol.3. 40.2 Different pH Values The different buffers were prepared at different pH values (3. The numerous advantages of the new set of esterases compared to commonly used esterases present a great economical potential to a suitable industrial partner in each of the application sectors. 36. 12. 30. flavor enhancer and animal feed moreover they can be used. The media supplements with either of 1% tween 20 or 0. 30. 20. 74 . fischeri. e. slightly modified) was used.1 Incubation Temperature Bioluminescence bacterium was grown on production medium and incubated at 24 hrs at different temperatures Viz: 10.3 Incubation Period The effect of incubation period was determined by incubating the production medium at different incubation periods viz. synthesis of carbohydrate derivatives. 2. 2.2 Spirit Blue Agar Broth culture was streaked on the spirit blue agar plates with substrate (Tween or Triacylglycerol). 0.6.025M MgSO4. Whole light organs were removed by dissection and homogenized in 700µl of sterile seawater (Ruby and Nagai. Material obtained in either of these ways was serially diluted in seawater complete (SWC) broth and the samples were spreaded on SWC (0. Then the plates were incubated at different temperature 6ºC. 30ºC for up to 15 days. 8mM KCl. The global market for industrial enzymes is constantly growing with a rate of 5-10% per year. 18. 7 and 9). 17ºC. The plates were observed after 6 hrs and every 12 hrs for the clearing of the blue or deep blue color around each streak. 0.2. 2% agar and 0. 2. edible oil (olive.3 Physical and Chemical Conditions for Esterase Production Different culture conditions were used to obtain the maximum levels of enzyme productivity by V. 2. food additives.g. feed processing detergents or detergent compositions. 6. 50 and 60ºC respectively.3.2. Experiments 2. Light organ fluid containing bacteria was obtained from pore that leads into channels with in the organ tissue. minus the diameter of the colony was considered to be proportional to the esterase activity rate. 6 Modern Applied Science Exterases have a wide range of industrial applications. paper and pulp. For detection of esterase activity the following basal medium (Zobell 2216E. wherever enantiopure compounds are needed and find application as research reagents in studies on plant cell wall structure. 0. 2. 1g of yeast extract. All carbon sources were sterilized separately (ethanol by filtration) and added to a final concentration of 2% (w/v). Information of lipolytic enzymes produced by marine Vibrio spp is particularly limited. cotton seed.

The elution containing esterase activity was pooled and used for further characterization. 2009 150µl of culture was inoculated into 200ml of sea water medium with substrate and incubated at 30ºC for 3 days.6M).7. electrophoresis performed by the method described by Lammeli et al. The portion was centrifuged at 20. The cell free filtrate was used as the crude enzyme for the purification experiments.4 Gel Filtration Chromatography (Sephadex G200) Esterolytic fractions collected from DEAE cellulose chromatography were redissolved in a small volume of 50mM phosphate buffer.1 Enzyme Production and Preparation of Cell Free Filtrate June.2 pH Optimum Enzyme assays were conducted at various pH in an emulsified reaction mixture containing 0. and further purified by gel filtration on Sephadex G200.1 ml enzyme.000 xg at 4ºC for 30 min.000 rpm for 30 min.0.2 Acid Precipitation The crude extract was precipitated by slowly adding 1N HCl at 4ºC with stirring until pH 4. For measurement of molecular mass of protein.4.7. 2.4. 2.3 Temperature Stability The solution of enzyme at the concentration of 45mg/ml was adjusted to pH 7. 2. 2.4 Temperature Optimum Enzyme activity was determined at various temperatures ranging from 5ºC to 65ºC.7. equilibrated with 50mM phosphate buffer. it was titrated to pH 9. fischeri in cell free broth.5 Enzyme Activity Assay Enzyme assay used for the determination of esterase activity upon emulsified substrate and Tween solution.5 to 10% of tween 20. Then 1 ml of the esterase solution in the buffer was added and the esterolytic reaction was observed for 40 min by titration as mentioned above. The supernatant was filtered using 0.5 Substrate and Enzyme Kinetics The esterase enzyme was incubated with various concentrations of substrate and the final substrate concentration ranged from 0.4 Purification of Esterase 2. Then the aliquots were assayed for activity.3 was attained.. equilibrated with 20 mM phosphate buffer. pH 7. commercial broad range molecular mass standard proteins were used. Control reaction mixture contained heat inactivated enzyme. Enzyme activity is expressed as U/ml and one unit (U) of activity is defined as µmols of free fatty acids liberated /min/ml by the enzyme solution under assay conditions. The pH was maintained by pH stat with 0. Elution with the same buffer was at the flow rate of 12ml/h. (1970) with some modifications (Vorderwulbecke et al.45 µm cellulose acetate filter units. The final concentration of Triacylglycerol (TAG) was 10-50mM.Cellulose) Dissolved pellet was first applied to a column of DEAE-Cellulose.6 Protein Mass Determination (Native PAGE) In order to study the undenatured protein profile of esterase enzyme from V. The reaction mixtures (except enzyme) were held at the respective temperature for 5 min before the addition of the enzyme. The proteins were eluted at a flow rate of 18 ml/h with a linear NaCl gradient (0.1 N HCl and aliquots were incubated at 30ºC for 4 hrs.8-20% (v/v).4. The mixture of the substrate and buffer (final volume 8ml) was adjusted to pH 8.4. The pellet was dissolved in 10mM phosphate buffer and brought buffer pH 7. 0.5 by the addition of 1N NaOH.02 NaOH and aliquots were incubated at temperatures ranging from 5ºC to 65ºC for 4 hrs. 2.0. 1992). 3 ml of fractions were collected. 2. Tween 0. Different concentrations of enzyme also studied in the concentration of 5.1N NaOH or 0.02 NaOH. The fractions containing strong esterase activity were pooled (fraction size of 3ml) and concentrate. 2. pH 7. pH 7. 2. The quantity of free fatty acids released was calculated from the total quantity of base used.Modern Applied Science 2.7.7 Characterization of Lipase Enzyme 2.5. Gel was casted by using discontinuous buffer system having 10% resolving gel and 5% stacking gel. 2. 15 and 20 microlitres. containing 0.1 pH Stability Enzyme solutions at a concentration of 70mg/ml were adjusted to various pH ranging from pH 2 to 11 with either 0.3 Ion Exchange Chromatography (DEAE. The precipitate containing the esterase activity was collected by centrifugation 10.7. 10. 4ml 50mM Tris.4 ml of Tween or TAG.1 to 0.. After incubation of the reaction mixture for 40 min at 30ºC.15M NaCl.0 with 0. Then aliquot was removed and assayed for activity. 75 . Protein bands were located by coomassie blue staining. 2.0 by 1N NaOH and the pH was maintained for 3 min by titration with 50mM NaOH solution (blank).

CaCl2 (0. unable to grow in the absence of NaCl. NaF (2mM). fischeri exhibited their maximum ability to biosynthesize esterase enzyme with in 48 hrs.bile salts.3. but not triolein. (2005).3 Carbon Source The ability of V.0) and in 0. 30 ml of a 2 days culture of esterase solution was sterilized by membrane filtration (0. In fact it actively splits tween 20 than tributyrin as good substrate. 76 . 3.2 pH This test found that the best buffer was phosphate buffer at optimum pH for the production of esterase enzyme was recorded at pH 7.3. Oils were not utilized by this strain.6 Enzyme Stability on Metal Ions and Other Chemicals Modern Applied Science Enzymes were preincubated for 1 h at 30ºC (pH 7. fischeri depended on the substrate utilized by the cells.Vol.1 Temperature From this study. KCl (2mM). The production level of esterase enzyme of V. fischeri was showed 36 mm of halos after 10 days incubation indicates that the strain V. tributyrin as a carbon source and energy material to produce esterase. fischeri bacteria at different temperatures. However.001M)..2 Screening of Esterase Enzyme In ZoBell modified media. (1995) found that esterolytic activity at moderate temperatures (eg. cyclic and sugar esters. sodium dodecyl sulphate (0. the temperature below or above the 30ºC caused a sharp decrease in enzyme yield as compared to the optimal temperature. the ammonic detergent. Results and Discussion 3. fischeri strains tested on VHA was small (2 to 5mm) dark blue green colonies. CuO2 (2mM).04% to prevent bacterial growth.. the size of halos increase after 3 days but the area of hydrolysis was smaller than that at 30ºC. and 20ºC.22µm. 8. FeSO4 (0.30ºC) was high with structurally diverse ester substrates including aliphatic.5% EDTA). They were halophiles. The esterase activity in the stored samples was determined periodically by the pH stat technique and expressed as percent initial activity. 6 2. which did not contain CaCl2 (except in test sample). the activity began after 12 hrs. 3. The similar result was observed by Shabtai and Mishne (1992). 3. salinity and pH have been characterized by Encarnacion et al. Assay was performed with the mixture. several lipolytic enzymes active and stable in extreme conditions of temperature. The habitats of this squid species must receive a significant input of cells of symbiotic V. In contrast. At 30ºC.. The specific production level of the enzyme examined was very low when ethanol serves as the sole carbon source. The colony morphology of the luminous V. Total viable luminous count range was varied from 4 to 18 CFU/ml. tributyrin) at 30ºC. observed that 30ºC was generally more favorable for esterase production. More over the use of sorbitan monoesters (tween 20) increased the esterase enzyme production. the expression of the enzyme was markedly enhanced. In spirit blue agar. Other chemicals tested were ethylene diamine tetra acetic acid (0. (1982) reported that the most strains of Pseudomonas sp. They produced yellow colonies on SWC agar plates.. The luminous V. 3. Interestingly V. At 17ºC. Millipore corp.). BaCl2 (0.5% SDS). More or less similar mm of halos as that in ZoBell modified agar was observed in spirit blue agar with the substrate (tween. 20ºC . Bruni et al. (1996) reported that the VHA media displays great potential as primary isolation medium and offers significant advantages over thiosulfate-citrate.1M Tris-HCl buffer with various ions and other chemicals (one at a time). NCMB 1082 was split all tweens.001M). 1994).7. (2007). The higher activity at 30ºC may be attributed to maximum growth of the organism and subsequently increased esterase secretion. Wood et al. In the dynamic field of biocatalysis. fischeri to utilizing tween 20.sucrose agar.0. Different substrates were used. The ions used were included NaCl (10mM). tributyrin. MnCl2 (2mM). 1.001M).001M).7. fischeri (Lee and Ruby. In order to favour the secretion of extracellur lipolytic enzymes the effect of temperature and carbon source has been studied by Dominguez et al. 9 strains showed good activity on water soluble tweens.3 Physical and Chemical Conditions for Esterase Production 3.3. fischeri isolate was motile Gram negative rods. A notable decline in the enzyme productivity occurred at both higher and lower pH values. The addition of fatty acids did not repress the production of the enzyme. Apparently low activity was observed at 6ºC. the V. but the maximum esterase productivity was attained in the tween 20.1 Bacteria and Growth Condition Vibrio fischeri encountered 100% of luminous bacteria. 4 on tween 85.7 Enzyme Stability During Storage Enzyme solutions from 2 days culture were stored at -20. with the water immiscible glycerol and triglycerides such as tributyrin.. the width of hydrolysis areas were measured to observe the esterase activity of luminous V. MgCl2 (0. Formaldihyde was added to a final concentration of 0. 3.001M). Harris et al. 3. 2. fischeri showed significant esterase activity. No.. the esterase activity of strain was appeared after 7 hrs with a large discoloration area or dark blue halos and the widest area observed after 1 day. SrCl2 (0.

0 and 30-40ºC respectively.3.4 Purification of Esterase 1N HCl precipitate of crude extract of cell free supernatant eluted in DEAE-Cellulose chromatography.. plantarum (Gobbetti et al. The temperature range was 30 to 90ºC. Bendikiene et 77 .. Purification of the esterase was performed in three steps of anion exchange chromatography and finally by gel filteration chromatography.2 pH and Temperature Stability The enzyme was stable over a wide pH range. 3. As indicated in figure 4. The concentration above 8 % reduced 50 % of the enzyme activity.Modern Applied Science 3. Smacchi et al. Kakariari et al. 2001).7 mM using pNPC6. fischeri was monomeric protein. 90% activity at 35ºC. (1999) assayed the esterase activity in the range of substrate concentration from 0. Smacchi et al. but during the stationary phase.3. Separation on Sephacryl S-300 gave a symmetrical single peak (Kakariari et al. (2000) obtained only one band with the crude cell free extract in SDS-PAGE. Tomioka (1983) estimated that the molecular weight of esterase was to be 36. significantly different from the complex esterase system of the P.000 by sodium dodecyl sulfate polyacrylamide. tributyrin and exhibited high activity only on the compound of sorbitan monoester.. This esterase had substrate specificity on tween 20. Sixty percentage of the activity available at 37ºC. In contrast Kakariari et al. (2000) stated that the extracellular esterase of A. In this pH the esterase enzyme was retained 100% of the optimum activity.6. Sephadex G150. Gel filtration (Sephadox G200) chromatogram shown in figure 2. 3. The esterolytic fractions were assayed in chromogenic agar plate with the substrate of tween 20 showed yellow zone around the active fractions..4 Incubation Period Highest enzyme production was observed in the incubation period of 72 hrs at 30ºC. suggesting that the enzyme is a monomer (Smacchi et al.0 and 30ºC. (2005) determined that the optimal pH for Est E1 esterase protein at various pH values of pH 3.5 Protein Mass The esterase from V. 3. 3. activity slowly decreased. Macarie et al. fischeri was showed molecular mass of 37 KDa. The temperature above 30ºC. phenyl sepharose and diethyl – (2-hydroxypropyl) aminoethyl column chromatography and by subsequent preparative polyacrylamide gel electrophoresis. 3.5.6. freudenreichii ssp freudenreichii strains described by Dupuis and Boyaval (1993). 2009 Esterase from the V. The esterase activity was represented by fractions between 28-34.. At the temperatures below 25ºC.. 3. Similarly crude preparations of esterases from lactic acid bacteria (Khalid and Marth. 1990) and purified esterase from L. Temperature Optima Vibrio fischeri esterase had an optimum pH of 7. More than 2mM inhibit the esterase activity. it exhibited a high activity only on compounds having both polyoxyethylene and fatty acyl moieties such as tweens (Tomioka. the enzyme had a temperature optimum of 30ºC. nicotianae 9458 had pH and temperature optima of 7.6. 1997) had pH and temperature 6.5 to 8 % of tween.6 Characterization of Esterase 3. 2000). as assess in the intracellular extract during growth.3 to 2.. Tomioka (1983) reported that tween-hydrolyzing esterase of Mycobacterium smegmatis was stable to heat treatment at 100ºC and to a wide range of pH. The purified esterase from gel filtration chromatography showed single peak corresponding to protein peak and exhibiting final specific activity of 300U/mg.. At pH below 4 or above 10 the loss of enzyme activity was appreciable. 2000).5 Time Course for Esterase Production June. reached maximum in the middle of the logarithmic phase and then decline rapidly. The pooled material was eluted in gel filtration (Sephadox G200) chromatography. In pH 6 retained 60% and pH 9 retained 70% activity. In native PAGE electrophresis it showed single band indicates esterase from V.5 on tween 20 substrate (Fig 3). The purified extracellular Arthrobacter nicotianae esterase showed a single band on SDS-PAGE corresponding to a molecular mass of about 32 kg mol-1.0 to 9. Similarly. (2000) reported that the production esterase.1 pH. respectively.3 Substrate and Enzyme Kinetic The esterase activity was reached at maximum in between the concentration of 0. 1983). the esterase activity was released in the culture medium (Choi and Lee. Gradual loss of activity with increased temperature up to 65ºC. The esterase had narrow substrate specificity. An esterase hydrolyzing tween 80 (polyoxyethylene sorbitan monooleate) was purified from sonicated cell lysates of Mycobacterium smegmatis ATCC 14468 by DEAE-Cellulose. fischeri produced maximum activity in stationary phase (Fig 1). at lower temperature the enzyme still showed activity. (2000) reported that the chromatography in both DEAE-cellulose and on Sepharose 6B resolved the esterase activity into one peak corresponding to the major protein peak. 70% of the activity retained at 40 and 45ºC. 30% actively retained at 50ºC. the maximal Lactobacillus casei esterase production was obtained intracellularly during the late logarithmic phase. indicating a simple enzyme system.. Rhee et al.5-7.

ethylene diamine tetraacetate (EDTA) or sulfhydryl inhibitors. Int. Evidently even the low concentration of NAF and CuO2 highly retarded the easterase activity.. W. Chelators and Other Chemicals on Esterase Figure 5 showed the results on the effect of various inhibitors on the activity of the esterase. Stimulation of novel thermostable extracellular lipolytic enzymes in cultures of Thermus sp. B.. which were showed no effect on enzyme activity. V. A. (2001). However with the realization of the biocatalytic potential of microbial lipases and esterases in the last one and a half decades. B.Vol. D. M. Microorganisms for industrial enzymes and Biocontrol. 40: 187-194. It produced 67% of the activity in the presence of EDTA. M. Biologija. J. References Bendikiene. Lait. CaCl2. & Sanroman. In this study the SDS was highly favored the activity of esterase (133%). K. 3. Georfalaki. Harris. 3. Maugeri. E. & Lee. The inhibitory action of former was stronger than that of latter. & Corsetti. 73: 2669-2684. H.. H. Similarly Moskowitz et al. J. Purification and characterization of an intracellular esterase from Propionibacterium freudenreichii ssp freudenreichii ITG 14. Hendrie. 67: 113-119.. J. Cristina. Surinenaite. & Antonio. 3. & Smith.4 Effect of Metal Ions. other metal ions except Cu2+. EDP Sciences. V. Lee. Khalid. A.. E. Dominguez. L. 60: 1565-1571. Lipolytic enzymes from extremophilic microorganisms.V. Smacchi.. Microbiol. M. Microbiolog. & Ruby.. Fe2+.. F. S. Owens. 62(9): 3548-3550. J. Effect of squid host on the abundance and distribution of symbiotic V. Lipolytic activity of marine bacteria influence of NaCl and MgCl2. M. Pastrana. P. & Boyaval. Kalantzopoulos. (2000) showed that the esterase from P. 9: 25-43. I. 1970). industrial fronts have shifted towards utilizing lipase. 24: 59-63. Fucinos. L. M. fischeri in nature. 78 ... A. L.. (2005). S. (1997).T & Alonzo. A selective and differential medium for Vibrio harveyi. (1990). Environ... Kakariari. E. C. 6 Modern Applied Science al. A linear relationship was also evident when enzyme activity was measured as a function of protein concentration. M. Rua. Appl.. L. V. Enzyme mediated relations are attractive alternatives to tedious and expensive chemical methods. S.13-21. & Juodka. The identification. These lipolytic enzymes stability suggests that the accelerated destruction of enzyme at higher temperatures.. No. E. Bioprocess and Biosystems Engineering. Dairy. Gen. Sara. & Shewan. M. & Marth. Choi. Culture conditions for the production of esterase from Lactobacillus casei CL 96. A. Microbiol. taxonomy and classification of luminous bacteria.. The same direct proportionality was found when crude extract were used as the source of enzyme (Oterholm et al. B. N.1: 27-30. J. Y. All other ions tested in the present study showed inhibition with relative degree of variation. (1996). Conclusion The demand for industrial enzymes particularly of microbial origin is ever increasing owing to their applications in a wide variety of processes. (2005) found the tween 85 was the best substrate among all the detergents studied and 10% concentration was optimal for the hydrolysis by lipolytic enzyme from Pseudomonas mendocina 3121-1. Except NaCl. The esterase enzyme activity was not inhibited by the EDTA. Environ.. G. Marine Biology. Frozen storage did not affect the enzyme activity to any greater extent. 491-501. Marcinkevieiene. Encarnacion. 73: 345-356. (1970). Esterase activity of dairy Propionbacterium.5 Enzyme Stability During Storage Figure 6 showed the esterase activity as a function of storage time at different temperatures. esterase enzymes for a variety of reactions of immense importance. P. Enzymes and Microbial Technol. Hodgkiss. M. (1993). Hg+. L. In the above scenario enzymes such as proteases and amylases have dominated the world market owing to their hydrolytic reactions for proteins and carbohydrates. Gobbetti. (1994). P. (2005). Longo. Purification and characterization of a cell surface-associated esterase from Lactobacillus fermentum DT41. Hydrolysis. E. & Tsakalidou. 64: 151-169. freudenreichii ssp freudenreichii did not affected by EDTA.6.. App. M. Kakariari et al. (1977) reported that the esterase of Mucor miehei was relatively unaffected by the high concentration of various salts. The specificity of Pseudomonas mendocina 3121-1 lipase. (1982). Dupuis.6. (2000). Bachmatova. Action of these two compounds could be attributed to their effect in creating the imbalance of ions in the reaction mixture by absorption or release.. Dairy Sci. (2007). respectively. H. Lactobacilli-their enzymes and protein ripening and spoilage of cheese: a review. G. Bruni.

. & Baratti. G. L. L. Microbiol. G. Oterholm. 2009 Macarie. Kim. (1970).. P. D. P. 59: 174-180. A. Symbiotic association of Photobacterium fischeri with the marine luminous fish Monocentris japonica: a model of symbiosis based on bacterial studies. O.2 3 2.. 3. App. & Erdman.. purification and properties of lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments. T. App.174: 4865-4870. (1977). Weat. (1999). & Witter..12 Activity (U) OD (650 nm) 0. A.5 1 0.97 3. Rhee. purification and properties of extracellular carboxyl esterases from Bacillus subtilis NRRL 365. Microbiol. J. Lait 80: 255-265. F.. Reichelt. Technol. Y. Gobbetti. Comparison of lipases by different assays. Dairy Sci. G... 71: 817-825.. E. Bacteriol. D. Sabu. Purification and properties of a glycerol ester hydrolase (lipase) from Propionibacterium shermanii. E. & Feldman.28 0.5 3. K. J. J. Biochem.Modern Applied Science June. (2000). M.. Ward. J. Margo. Z. J. Y.18 3. Bull. Production.155: 1249-1259. E. Characterization of a novel thermostable esterase as an industrial catalyst. A. Biotechno. App. Fernandez. Purification and characterization of the Tween. Smacchi. (1995). (2003). 94: 283-330. & Cowan.. Arch.49 0. N.hydrolyzing esterase of Mycobacterium smegmatis. (1976). Biochem.43 0. L. Microbiolog. A.18 3.5 2 1. Kieslich. (1973). Purification and characterization of an extracellular esterase from Arthrobacter nicotiancae 9458. P. H. Shen. A. App. (1990). T. New thermophilic and thermostable esterase with sequence similarity to the hormone – sensitive lipase family.3 2. J. G.1 0. A squid that glows in the night: development of an animal bacterial animal mutualism. J. & Mishne. Production. R. Ordal. R. & Nagai. H. (1992). Sources. luminous bacteria.. Enzyme Microb. Meghji. J. N. J. R. J. K. L. & Araujo.15 0. 63: 1865-1870. Biotechnol. G. & Oh. & Nealson. M. & Baumann. Taxonomy of the marine. Properties of the esterase produced by Mucormichei to develop flavor in dairy products. Ruby. Environ.41 0. I.36 0. 60: 1260-65. Indian J.08 0. H. 21: 313-322. Biol. Microbial.69 1. Biosci. Ruby. Characterzation of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis. (1992). Biotechnol. Cassaigne. W.4 0. Shabtai. 20: 16-22. Microbiol. D. J. (2005). 2: 334-341.5 0 10 20 30 40 50 60 70 80 90 100 110 0. K. 14: 631-639. Ahn. Moskowitz.2 Time (Hours) Figure 1. & Fox. (1992).89 2. P.24 0. E.04 0. M. D. & App. A. Rossi. Esterase Production during Growth of V. cloned from a metagenomic library. Tomioka. Environ. Vorderwulbecke. Environ. V. 151: 574-586. fischeri 79 . A. 56: 3735-3740. Bacteriol. Wood. (1983). K. properties and applications of microbial therapeutic enzymes.7 2.

2 1 0. 3. Elution Profile of Esterase from the Sephadex G200 Column 120 100 ESTERASE 80 Activity (%) 60 40 20 0 0 -20 pH 2 3 4 5 6 7 7. No. fischeri 80 .5 8 9 10 11 Figure 3. 6 Modern Applied Science 1.4 0.Vol.6 Active Fraction OD Absorbance of protein (280 nm) 0.8 0.2 0 24 12 16 20 28 32 36 40 44 52 48 56 60 0 4 8 Fraction num ber Figure 2. Effect of pH on the Esterase from V.

fischeri 140 133 120 100 100 100 100 Zone (%) 80 80 75 75 75 67 75 67 ESTERASE 60 42 40 20 0 0 N aC l Ba C l M 2 gC l2 0 0 K Cl Fe So 4 Ca C l2 Sr Cl 2 N aF M nC l2 Cu O 2 SD ED S TA Co nt ro l Metal ions & inhibitors Figure 5. 2009 100 ESTERASE 80 Activity (%) 60 40 20 0 0 -20 Tem perature 5 10 15 20 25 30 35 40 45 50 55 60 65 Figure 4. Effect of inhibitors on Esterase enzyme 81 .Modern Applied Science 120 June. Effect of Temperature on the Esterase from V.

3. fischeri Esterase at Different Temperature 82 . Storage Stability of V.Vol. 6 120 Modern Applied Science 100 20ºC 8ºC 1ºC 80 Activity (%) -20ºC 60 40 20 0 0 20 40 60 80 100 120 Days Figure 6. No.

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